It is widely known that platelets closely participate in crisis of so-called thrombosis represented by myocardial infarction and cerebral thrombosis ("Platelets", edited by Yamanaka and Yamazaki, Igaku-Syoin, pp. 158-163 (1991)). Recently, it has been reported that the binding between von Willebrand factor as one of blood proteins and glycoprotein Ib located on platelet surfaces is important for platelets to adhere to intravascular subendotherial tissue, which is considered as an early reaction to cause thrombosis (J. P. Cean et al., J. Lab. Clin. Med., 87, 586-596 (1976)).
It is known that the binding between the two species of the proteins does not occur in an ordinary state, but it occurs only when a high shear stress is exerted in vivo (T. T. Vincent et al., Blood, 65, 823-831 (1985)). The methodology to observe the binding exo-vivo includes a widely spread method which uses certain substances such as ristocetin as an antibiotic (M. A. Howard, B. G. Firkin, Thromb. Haemostatis, 26, 362-369 (1971)) and botrocetin as a protein originating from a snake venom (M. S. Read et al., Proc. Natl. Acad. Sci. U.S.A., 75, 4514-4518 (1978)). Platelet aggregation occurs when these substances are added to a suspension of platelets. These aggregation depends on the binding between von Willebrand factor and glycoprotein Ib (M. A. Howard, B. G. Firkin, M. S. Read et al., supra).
Several compounds have been already reported, which exhibit an inhibiting action on the platelet aggregation mediated by ristocetin or botrocetin. Such known compounds include, for example, aurintricarboxylic acid (M. D. Phillips et al., Blood, 72, 1989-1903 (1988)), and dye substances such as aromatic amidino compounds (J. D. Geratz et al., Thromb. Haemostasis, 39, 411-425 (1978)), as well as partial fragment peptides of von Willebrand factor or glycoprotein Ib (Y. Fujimura et al., J. Biol. Chem., 261, 381-385 (1986); K. Titani et al., Proc. Natl. Acad. Sci. U.S.A., 84, 5610-5614 (1987)).
It has been also reported that peptides having similar platelet aggregation-inhibiting activities are present in snake venoms. An international publication pamphlet of WO9208472 describes such peptides from Crotalus horridus horridus and Cerastes cerastes, each peptide comprising two different strands having a molecular weight of about 25 kilodaltons in which the homology is high at least in amino acid sequences on N-terminal side. A platelet aggregation-inhibiting peptide, which has been reported by Peng et al. (M. Peng et al., Blood, 81, 2321-2328 (1993)) as obtained from Echis carinatus, is also extremely similar to the peptides described above in its in vitro activity, molecular weight, etc. Any of the platelet aggregation-inhibiting peptides originating from snake venoms inhibits platelet aggregation mediated by ristocetin or botrocetin at a low concentration of 2 to 5 .mu.g/ml or less in vitro.
The international publication pamphlet of WO9208472 does not describes the anti-thrombus activity upon administration to animals. Accordingly, as described in Example 1 in this specification, the present inventors purified a peptide having equivalent properties to the peptide described in the pamphlet, considering a purification method for the peptide originating from Crotalus horridus horridus described in the pamphlet, in order to investigate the action upon administration to animals. As a result, the present inventors observed almost complete disappearance of platelets in blood upon administration in a small amount of 100 .mu.g/kg. However, the international publication pamphlet of WO9208472 describes neither suggestion nor solution of such a problem upon administration to animals.
Moreover, the peptide from Echis carinatus obtained by Peng et al. has its molecular weight of about 26 kilodaltons under a non-reduced condition as measured by SDS-polyacrylamide gel electrophoresis, while the peptide provides two peptides of about 14 kilodaltons and about 16 kilodaltons under a reduced condition. Accordingly, it is postulated that this peptide is also homologous to the peptide originating from Crotalus horridus horridus. It has been also reported for this peptide that remarkable decrease in platelets is observed upon administration to animals (M. Peng et al., Blood, 81, 2321-2328 (1993)).
The respective peptides originating from snake venoms, which inhibit the binding between von Willebrand factor and platelets, have high homology in their amino acid sequences, and they are also extremely similar in their molecular weights. In addition, according to the result described above, it is assumed that these peptides also have an activity to cause decrease in platelets in vivo. Namely, these peptides inhibit the binding of von Willebrand factor with platelets in vitro at a low concentration, however, they are difficult to be utilized as an anti-thrombosis drug for in vivo administration. The present inventors have considered the mechanism to cause the decrease in platelets as follows.
The peptides from snake venoms, which inhibit platelet aggregation mediated by ristocetin or botrocetin at a low concentration, have, for example, an amino acid sequence as described in the international publication pamphlet of WO9208472. According to the amino acid sequence, the peptide from snake venom conserves approximate positions of cysteine residues and a part of a sequence considered to be necessary for the lectin activity (W. I. Weis et al., Science, 254, 1608-1615 (1991)), as compared with a peptide having a calcium-dependent lectin activity reported by Drickamer et al. and referred to as "C-type lectin" (K. Drickamer, J. Biol. Chem., 263, 9557-9560 (1988)).
Other than the above, peptides having high homology to the C-type lectin in their amino acid sequences have been obtained from snake venoms, including, for example, botrocetin obtained from Botrops jararaca (Y. Usami et al., Proc. Natl. Acad. Sci. U.S.A., 90, 928-932 (1993)), rattle snake lectin obtained from Crotalus atrox (J. Hirabayashi et al., J. Biol. Chem., 266, 2320-2326 (1991)), and alboaggregin obtained from Trimeresurus albolabris (E. Yoshida et al., Biochem. Biophys. Res. Commun., 191, 1386-1392 (1993)).
These peptides also conserve approximate positions of cysteine residues and a part of the sequence considered to be necessary for the lectin activity, as compared with C-type lectin. It is also known that C-type lectin has an activity to agglutinate cells, bacteria and so on through glycoproteins (sugar chains) on their cell membranes (K. Drickamer, J. Hirabayashi et al., supra).
When the platelet-aggregation is measured in vitro, collected blood samples are often added with citric acid, sodium citrate, and ethylenediaminetetraacetic acid (EDTA) as anticoagulants, and hence calcium is chelated in such a platelet aggregation-measuring system. Accordingly, calcium-dependent reactions are inhibited. For this reason, it is difficult to detect in vitro whether or not the peptides from snake venoms exhibiting the platelet aggregation-inhibiting activity have the lectin activity.
Thus it is considered that the peptides from snake venoms express the C-type lectin activity in the presence of calcium in vivo upon administration to animals, and they cause aggregation of platelets. It is assumed that such an action is one of factors to cause the observed phenomena such as collection of platelet aggregates in microvessels, and consequent decrease in platelets or thrombocytes (thrombocytopenia).
Therefore, in order that the peptide from snake venom which inhibits the binding of von Willebrand factor with platelets functions as an anti-thrombosis drug which is efficacious in vivo such as in experiments with animals, it is necessary to obtain a new active peptide prepared by conversion into a molecule which does not cause decrease in platelets.